Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide-PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide-PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.
Cohesive ends of 16-3, a temperate phage of Rhizobium meliloti 41, have been identified as 10-base-long, 3-protruding complementary G/C-rich sequences. terS and terL encode the two subunits of 16-3 terminase. Significant homologies were detected among the terminase subunits of phage 16-3 and other phages from various ecosystems.The terminase enzyme is part of a large nucleoprotein complex that packages viral DNA into the capsid (4, 16). It has been well studied in the case of phage , but quite a few other members have been identified in different phages and studied in detail (6). Studying the packaging reaction of new phages provides the opportunity to identify alternative mechanisms and can extend our understanding of the packaging process and the formation and functioning of nucleoprotein complexes in general. Studies of similar functions in diverse systems are also required to understand the evolution of complex biological machines.Phage 16-3 is a temperate phage of Rhizobium meliloti 41. The genetic and physical maps of the phage have been established previously (9,11,13,22) and summarized in reference 7. Genes, proteins, and chromosomal sites for several functions of the phage have been studied in more detail. These include (i) the main repressor protein, C, required for establishing and maintaining lysogeny, and the operator regions where the C protein binds (8,10,25); (ii) the components of the sitespecific recombination system (5,12,23,29,30); (iii) the immX regulatory region conferring immunity against homoimmune phages (7); and (iv) the recently identified h gene encoding the tail fiber protein (26).Identification of the cohesive ends of phage 16-3. The plasmids used during the course of the study are listed in Table 1. The 96BglII-14EcoRI fragment ( Fig. 1) (numbering was based on the map of restriction sites in reference 13) of phage 16-3 was subcloned from cosmid pDH31 (11), which contains the region where the covalently ligated ends of the phage chromosome are located. The resulting plasmid, pPAG165, was used to determine the nucleotide sequence of the entire fragment. Nucleotide sequence determination was performed by the dideoxy chain termination method (28) with the fmol DNA cycle sequencing system (Promega). Primer 1, 5Ј-CACGGCTTCGGCGGCGC TGTC-3Ј, and primer 2, 5Ј-GGCAAGAAGGTCGTGACCTA TG-3Ј, were designed close to the expected ends, and the isolated 92EcoRI-cos right and cos left -14EcoRI fragments from phage DNA were used as templates for a pair of sequencing reactions to distinguish between 5Ј and 3Ј overhangs (Fig. 2). A 10-bp sequence region existing in pPAG165 was absent from the sequences of both end-fragments, hence we concluded that the chromosome of phage 16-3 ended in 10-base-long, 3Ј-protruding, single-stranded, complementary sequences (5Ј-. . .CCG-GCGTCGG-3Ј and 3Ј-GGCCGCAGCC. . .-5Ј). The cos sequence has high G/C content and shows dyad symmetry. The symmetry can be recognized in patches within the duplex DNA flanking the single-stranded ends (Fig. 1).Identification of genes around...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.